Materials and methods

The participants of the clinical trials included 5-year students of the dentistry department of the SMANN, clinical residents from the chair of restorative dentistry and volunteers – all together 18 persons at the age of 21, all of them being practically healthy. The epidemiological survey encompassed determination of caries intensity by the CPR index, constants “C”, “P”, “R” (caries, pulpitis, removed teeth) and the hygienic index (HI) according to Green -Vermillion. The condition of the parodontium was determined by the Schiller-Pisarev assay as well as by the papillary-marginal-alveolar index (PMA). The condition of the local immunity of the mouth cavity was determined by the study of the amounts of natural colonization of buccal epithelium of the mouth cavity mucous and by the coefficient of the local immunity factors balance.

All the volunteers were divided into 2 arms:

1st, or the ELIXIR group – the mouth hygiene was maintained by the new anti-cavity mouthwash both domicilliary and in the hygienist room of the dental clinic of the SMANN.

2d, or the control group of the students maintained their mouth hygiene by traditional means.

All the participants were using tooth paste “Zhemchug” to clean their teeth 2 times a day – after breakfast and before going to bed – for 3-5 minutes during a month. They also rinsed their mouth cavities with the 20-25 ml of the new anti-cavity mouthwash. The CPR index, HI according to Green-Vermillion and the papillary-marginal-alveolar index (PMA) were determined and the Schiller-Pisarev assay was taken for each participant before and after administration of the anti-cavity mouthwash. The index of colonization of buccal epithelium (ICBE) and the coefficient of the local immunity factors balance of the mouth cavity were also determined.

The index of colonization of buccal epithelium (ICBE) by microorganisms was determined for 100 patients at the age of 21. The buccal epithelial cells were received from the donors before and after administration of the anti-cavity mouthwash. The level of natural colonization was calculated by the number of the attached bacterial cells on one epithelial cell. Epithelial cells of almost healthy participants with a different degree of cariosity intensity and hygienic conditions of the mouth cavity who were maintaining mouth hygiene with and without the anti-cavity mouthwash (by traditional means) were used for the study. Epithelial cells were taken with a sterile curette from the inner surface of the cheek and placed into a buffered saline solution. Then it was three times centrifuged to wash off the irrelevant bacteria and a suspension was prepared. To study the natural colonization a buccal epithelium swab was taken, then fixed and colored according to the Romanovsky-Gimse method to examine under a microscope. 100 epithelial cells were examined. Each epithelial cell separately was subject to thorough examination. Calculation of the average number of the attached bacterial cells on each epithelial cell was conducted. The number of the bacterial cells was expressed in balls according to a developed scale: (under 9 bacterial cells – 0 balls, 10-59 bacterial cells – 1-2 balls, 60-89 bacterial cells – 3 balls, 90-119 bacterial cells – 4 balls, 120-159 bacterial cells – 5 balls and 160 and more bacterial cells on one epithelial cell – 6-11 balls).

Evaluation of the coefficient of local immunity factors balance in the mouth cavity.

To determine the level of mouth cavity immunity, it was necessary to determine the level of secretory immunoglobulin A (sIgA), serum IgA and IgG as well as the coefficient of local immunity factors balance.

The integrated index encompasses a lot of characteristics of the mouth cavity immunity – the amount of saliva serum immunoglobulins A,G and lysozym in particular. Immunoglobulins and lysozym activity were determined in combined saliva which was taken at a particular time – in the morning before breakfast by spitting in a sterile glass tube 5-7 ml of saliva without stimulating salivary glands. The tube with the mouth liquid was closely plugged by a sterile cotton ball and was attributed a number according to the list. It was frozen and stored in a vertical position. The total number of the tests equals 64.

Determination of secretory immunoglobulin A (sIgA).

Quantiative estimation of sIgA in the mouth liquid was performed by the method of radial immunodiffusion (RID) (developed by O.Manchini and A.Carbonara in 1965 and modified by E.V.Chernokhvostikova and S.I. Golderman in 1975). Antiserum versus secretory component (sc) and the corresponding manufacturing standard of Moscow I.I.Mechnikov research & development institute were used for the tests. Secretory immunoglobulin A was determined in the supernatant fluid and its concentration was conveyed in g/l. 64 tests were performed all together.

Determination of serum immunoglobulins A and C (IgA, IgC).

Quantitative evaluation of serum immunoglobulins A (IgA) and C (IgC) in saliva was performed by the method of radial immunodiffusion (RID) (developed by O.Manchini and A.Carbonara in 1965 and modified by E.V.Chernokhvostikova and S.I. Golderman in 1975). Monospecific antiserums (manufacturing standard of Gorky research & development institute) were used for the tests. IgA and IgC were determined in the supernatant fluid and the concentration was conveyed in g/l. 64 tests were performed all together.

Determination of lysozym activity.

Lysozym activity was determined in combined saliva (%) which was taken at a particular time – in the morning before breakfast by spitting in a sterile glass tube 5-7 ml of saliva without stimulating salivary glands. Lysozym activity of combined saliva was determined by the photonephelometric method of V.G. Dorofeichuk (1968). The method is based on the lysozym’s ability to lyze cell wall mucopolysaccharides of the standard strain Micrococolysodeikticus. A phosphate buffer suspension pH = 7.2 – 7.4 was prepared out of the test isolate m. Lysodeikticus. Then it was filtered and standardized with photoelectric colorimeter – 56 (PEC-56) using a green light filter ( the working wavelength was 540 nm) in a tray with the working length of 3 mm. At the nephelometry the level of light transmittance of the suspension was increased to 20% (4 bln bacteria). 0.03 of the medium under examination was added to 1.47 ml of the prepared microbial suspension. The tubes were kept at the temperature of + 37 °C for 60 minutes and then nephelometry was conducted under the same conditions which were maintained at the standardization of the original suspension. To determine the percentage of lysozym activity the percentage of the original microbial suspension light transmittance (20%) was deduced from the test suspension light transmittance percentage. The test saliva was diluted with phosphate buffer 1:20. 64 tests of lysozym concentration in mouth liquid of patients with different degrees of mouth cavity condition were conducted.

Determination of the mouth cavity immunity factors balance.

The total number of IgA tests equals 64, of IgC tests – 64 and of lysozym tests – 64. After that the coefficient of the mouth cavity immunity factors balance was determined for 64 patients. The coefficient of the mouth cavity immunity factors balance, being a complex index of the local mouth cavity immunity conditions (N.I.Tolmachiova, 1987), allows to show these conditions mathematically. There being a lot of characteristics of local immunity interacting with each other, it is necessary to be able to convey the balance of the values under examination mathematically. So, the coefficient of the mouth cavity immunity factors balance is exactly the complex index we need. The formula for its determination is designed on the basis of functional liaisons of lysozym with immunoglobulins:

IgA and IgG – immunoglobulin concentration (g/l)

Liz – activity of combined saliva lysozym (%)

40% - a tentative norm of lysozym activity

0.6 is the ratio IgA/IgG which was registered with a prevailing majority of healthy children.

According to our data with the coefficient of the mouth cavity immunity factors balance equaling to 0.1-1.0 patients may be referred to as healthy; 1.1-2.0 – high risk group; patients with the coefficient of 2.1 and more – may be referred to as unhealthy with an unfavorable condition of the mouth cavity immunity.

Statistics.

Analysis of the study results was performed with the method of mathematical statistics. Dependability of the average values’ differences was proceeded by the Student criteria.